The ATP-binding cassette (ABC) transporter superfamiiy contains membrane proteins that translocate a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Overexpression of certain ABC transporters occurs in cancer cell lines and tumors that are multidrug resistant Genetic variation in. these genes is the cause or contributor to a wide variety of human disorders with Mendelian and complex inheritance including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response phenotypes. Comparison of the human ABC superfamily to that of other sequenced eukaryotes including Drosophila indicated that there is a rapid rate of birth and death of ABC genes and that most members carry out highly specific functions that are not conserved across distantly related phyla.
The ABC transporter family includes cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as transporter or channel for chloride ions. Chloride is the predominant physiological anion; therefore, Cl− channels play critical roles in cell physiology. Plasma membrane Cl− channels are crucial to the process of secretion in many epithelial tissues such as the kidney, the intestine, and the airway. Plasma membrane Cl− channels are involved in cell volume regulation in a wide variety of cells. Intracellular Cl− channels play important roles as anion shunt pathways for endosomal acidification. A Cl− channel gene is the locus of the primary defect in several human diseases; including Bartter's syndrome, Dent's disease, myotonia, and some forms of epilepsy; Cl− channel proteins play important roles in a variety of other conditions, including cancer. Despite their central roles in many physiological processes, our understanding of the structures and mechanisms of anion-permeable channels has lagged far behind that of their cation-permeable peers. One clear reason for this discrepancy is a paucity of specific probes that may be useful as tools for studying the permeation pathways and/or gating mechanisms, of Cl− channels. Indeed, the Cl− channel blockers available at present work with very low affinity and poor specificity.
Venoms from snakes, scorpions, marine snails, and spiders are rich sources of peptide ligands that have proven to be of great value in the functional exploration of cation channels . Peptide ligands have proven to be among the most potent and selective antagonists available for voltage-gated channels permeable to K+, Na+, and Ca2+, and have been very useful tools for detailed structural analysis of these proteins. Pore-blocking toxins provide clues about the arrangement of channel domains, about the interactions between the permeant ions and the pore, and about the proximity and interactions of the gating machinery with the pore. Gating modifiers provide tools to dissect the processes underlying the transitions between gating states. Peptide ligands have high potential as lead compounds for the development of therapeutics targeting pain, diabetes, multiple sclerosis, cardiovascular diseases, and cancer. Because peptide ligands have well-defined structures, constrained by disulfide bridges, they bind with much higher affinity and specificity than other blockers available to date, and report the structures of their targets at molecular detail. Unfortunately, although several potential chloride channel toxins have been identified, peptide inhibitors of chloride channels of an identified molecular target, including CFTR, have not been described.
In 1992, Strichartz and colleagues described the isolation of chlorotoxin (ClTx), a small basic peptide capable of inhibiting low-conductance Cl− channels from rat colon or brain reconstituted into lipid bilayers. However, neither recombinant ClTx, nor native ClTx isolated from venom, has been shown to block CFTR or any other Cl− channel of known molecular identity. Native ClTx also had no effect on Ca2+-activated Cl− channels or volume-regulated Cl− channels, when applied to the bath. Sontheimer and colleages have shown that ClTx inhibits the migration of glioma cells by inhibition of matrix metalloproteinase-2. A recent study suggests that recombinant ClTx inhibits an endogenous Ca2+-activated Cl− channel in astrocytes, but the molecular identity of this channel is also unknown.
Structure/function studies have led to improved understanding of which parts of the CFTR protein form the permeation pathway, and of the mechanism of binding and hydrolysis of ATP at the NBDs that regulate channel gating. However, little is known about how binding and hydrolysis of ATP controls the conformation of the pore to regulate transitions between open and closed states. The availability of a peptide that interacts with CFTR in a state-dependent manner will allow the application of quantitative approaches previously not accessible for answering these questions.
Therefore, it is an object to provide ABC transporter ligands and methods of their use.
It is another object to provide peptide compositions that interact with CFTR in a state-dependent manner.
It is another object to provide peptide compositions that block or inhibit Cl− channels.
It is yet another object to provide peptide compositions that block or inhibit Cl− channels for the manufacture of a medicament.
It is another object to provide pharmaceutical peptide ion channel blockers or inhibitors and methods of use thereof.
It is still another object to provide methods for treating ABC transporter-related disorders with peptide inhibitors of ion channels.